Abstract: :
Purpose: Cyclic GMP-gated cationic channels (CNGCs) are essentialfor phototransduction in the vertebrate retina. In chick retinalcones, the affinity of CNGCs for cGMP is under circadian control,and is higher during the subjective night than during the subjectiveday. This effect persists on the second day of constant darkness(DD) after entrainment to 12:12 hr LD cycles in cells maintainedeither in vivo or in vitro. Circadian modulation of cGMP affinityis driven by rhythms in the activity of the MAP kinase Erk,which is maximally active during the subjective night. Herewe examine possible steps that intervene between the photoreceptorcircadian oscillator and Erk.Methods: Properties of CNGCs weredetermined by inside-out patch clamp recordings from culturedchick retinal photoreceptors on the second day of DD after LD12:12 entrainment in vivo or in vitro. In vitro entrainmentoccurred in a culture chamber equipped with lights and timers.Embryonic chick cones were dissociated at E6, which resultsin a highly enriched population of cones, which contain largeoil droplets and rudimentary outer segments. Other endpointswere based on immunochemical assays of Erk and Ras activation.Results: Application of the cAMP protagonist forskolin, or thecAMP analog 8CPT-cAMP caused a significant (p < 0.05) increasein cGMP affinity of CNGCs when they are applied to cones duringthe subjective day (CT4-7) but not during the subjective night(CT16-19). Forskolin also stimulates cone Erk activity. Thiseffect is greater during the subjective day than the subjectivenight. Conversely, the adenylate cyclase inhibitor MDL12330Acaused a decrease in CNGC affinity (p < 0.05) when appliedat CT16-19, but had no effect at CT4-7. Dopamine and the D2agonist quinpirole, which also inhibit adenylate cyclase incones, caused a similar effect. A second potential mechanismfor Erk activation entails small GTPases such as Ras, Rac, andRap. Ras, in particular, causes activation of the protein kinaseRaf, leading to activation of MEK1 and stimulation of Erk. Wehave observed that Ras activity in cone photoreceptors is muchgreater during the subjective night (CT16-22) than the subjectiveday (CT4-10) in chick cones free-running on the second day ofDD.Conclusion: Cyclic AMP and Ras may contribute to circadiancontrol of the MAP kinase Erk, and modulation of cGMP-gatedchannels of cones.